Project Summary/Abstract Recent biologic and genetic studies implicate ubiquitination in the regulation of several inflammatory and cell survival pathways in inflammatory bowel diseases (IBD). IBD is manifested by mucosal ulceration, loss of the integrity of the intestinal epithelium, and inflammation of the gut mucosa. The events that lead to disruption of the intestinal barrier in IBD are incompletely understood. We have been studying a potent regulator of ubiquitin dependent signaling that is linked genetically and epigenetically to IBD, called A20 (also known as TNFAIP3). A20 is a negative regulator of NF?B that limits inflammation and restricts multiple cell death pathways. As death of intestinal epithelial cells (IECs) could compromise the intestinal barrier and contribute to IBD, in this proposal we focus on A20's role in preserving IEC survival to protect against IBD. During our investigations of A20, we observed that the expression of another ubiquitin-mediator, called ABIN-1, was increased in cells when A20 was deleted. ABIN-1 (also known as Tnip1) is also genetically linked to IBD and binds to A20. While A20 is a ubiquitin (Ub) editing enzyme, ABIN-1 binds poly-Ub chains. The functional relationship between A20 and ABIN-1 has never been studied in vivo. Our recent studies have unveiled a surprising and dramatic epistatic relationship between A20 and ABIN-1. These observations have led us to hypothesize that A20 and ABIN-1 protect against IBD by preserving survival of IECs, in addition to their ability to restrict immune cell activation. Accordingly, we propose to explore how A20 and ABIN-1 work together to maintain intestinal barrier function and homeostasis in a new mouse model in which A20 and ABIN-1 are inducibly and specifically deleted in intestinal epithelial cells. We will test how these proteins regulate cell autonomous survival pathways in intestinal organoid cultures. To understand the precise mechanisms by which A20 and ABIN-1 function, we have created several knock-in lines of mice with strategic point mutations that abrogate specific biochemical functions of A20 and ABIN-1. Using these new mouse strains, we will determine the biochemical functions of A20 and ABIN-1 that support IEC survival. The proposed studies will establish a new, inducible mouse model of IBD that will allow dissection of the physiological preservation of IEC homeostasis. The delineation of the ubiquitin controlled molecular pathways that lead to IEC death will yield mechanistic insights into how to intercede in the disease course to promote intestinal healing.